Recombinant DNA technology has been utilized to generate transgenic plants that express desirable phenotypic traits and protein products. However, the production of specialty chemicals such as fatty acids, amino acids, specialty plastic-like compounds, unique peptide hormones, etc. in seeds of plants often causes loss of seed viability and germination. This represents a major impediment to the development of biosynthetic strategies for producing commercially important products in plants because seed multiplication is essential for large-scale commercial production. The present invention is directed to the production of genetically engineered plants having a desired gene that is silent during seed multiplication but is capable of being activated to express its encoded gene product once sufficient numbers of transgenic plants have been generated.
The expression of a gene is generally directed by its own promoter, although other DNA regulatory elements are necessary for efficient expression of a gene product. Promoter sequence elements include the TATA box consensus sequence (TATAAT), which is usually 20 to 30 base pairs (bp) upstream of the transcription start site. In most instances the TATA box is required for accurate transcription initiation.
Promoters can be either constitutive or inducible. A constitutive promoter controls transcription of a gene at a constant rate during the life of a cell, whereas an inducible promoter's activity fluctuates as determined by the presence (or absence) of a specific inducer. The regulatory elements of an inducible promoter are usually located further upstream of the transcriptional start site than the TATA box. Ideally, for experimental purposes, an inducible promoter should possess each of the following properties: a low to nonexistent basal level of expression in the absence of inducer, a high level of expression in the presence of inducer, and an induction scheme that does not otherwise alter the physiology of the cell. The basal transcriptional activity of all promoters can be increased by the presence of “enhancer” sequences. Although the mechanism is unclear, certain defined enhancer regulatory sequences are known, to those familiar with the art, to increase a promoter's transcription rate when the sequence is brought in proximity to the promoter.
The creation of a transformed cell requires that the DNA be physically placed within the host cell. Current transformation procedures utilize a variety of techniques to introduce DNA into a cell. In one form of transformation, the DNA is microinjected directly into cells though the use of micropipettes. Alternatively, high velocity ballistics can be used to propel small DNA associated particles into the cell. In another form, the cell is permeablized by the presence of polyethylene glycol, thus allowing DNA to enter the cell through diffusion. DNA can also be introduced into a cell by fusing protoplasts with other entities which contain DNA. These entities include minicells, cells, lysosomes or other fusible lipid-surfaced bodies. Electroporation is also an accepted method for introducing DNA into a cell. In this technique, cells are subject to electrical impulses of high field strength which reversibly permeabilize biomembranes, allowing the entry of exogenous DNA sequences.
In addition to these “direct” transformation techniques, transformation can be performed via bacterial infection using Agrobacterium tumafaciens or Agrobacterium rhizogenes. These bacterial strains contain a plasmid (called Ti or Ri respectively) which is transmitted into plant cells after infection by Agrobacterium. One portion of the plasmid, named transferred DNA (T-DNA), is then integrated into the genomic DNA of the plant cell. This system has been extensively described in the literature and can be modified to introduce foreign genes and other DNA sequences into plant cells.
Transformed cells (those containing the DNA inserted into the host cell's DNA) can be selected from untransformed cells if a selectable marker (or visible marker) was included as part of the introduced DNA sequences. Selectable markers include genes that provide antibiotic resistance or herbicide resistance. Cells containing these genes are capable of surviving in the presence of antibiotic or herbicide concentrations that kill untransformed cells. Examples of selectable markers include the bar gene which provides resistance to the herbicide Basta, the nptII gene which confers kanamycin resistance and the hpt gene which confers hygromycin resistance. Visible marker genes express products that enable a visual identification of host cells, and thus allow for the identification of cells transformed with the visible marker gene.
Once a transformed plant cell is generated, an entire plant can be obtained through cell culturing techniques. Individual cultured cells divide to give rise to an undifferentiated mass of cells called callus tissue. Once callus tissue is formed, shoots and roots may be induced from the callus by techniques known to those familiar with the art, and the resulting plantlets can be planted. Alternatively, somatic embryo formation can be induced in the callus tissue. These somatic embryos germinate as natural embryos to form plants.
In accordance with the present invention the expression of commercially valuable products that have a detrimental impact on plant cell development is controlled to minimize the negative impact on the plant while maximizing the production of the product. The controlled expression of a desired gene product can be achieved by operably linking the coding sequence of the gene to an inducible promoter. In particular, inducible promoters can be utilized to achieve selective and timed gene expression in plants. For example, a chemically-induced gene promoter can be linked to a desired gene, and plant cells can be transformed with the promoter/gene construct to produce a transgenic plant. The transgenic plant can then be sprayed with the appropriate chemical to induce expression of the gene during the desired stage of development.
A strategy based on the use of environmentally controlled inducible promoters suffers from several disadvantages: 1) the limited number of possible promoters (e.g., heat shock promoter, copper inducible promoter), and 2) the problems associated with treating large numbers of plants in a natural environment. In addition, many inducible genes tend to be “leaky”, that is the promoters will express low levels of the gene product even in the absence of the inducer. The present invention eliminates the need for an external treatment to induce selective gene expression and provides and inexpensive approach to producing commercially valuable products in plants wherein the products have a detrimental impact on plant development.
The present invention utilizes site-specific recombinase systems (FLP/FRT, Cre/Lox, etc.) to control the expression of recombinant gene products in plants. In particular, a transgenic plant is generated that contains the desired gene, but the expression of the gene is blocked by a “blocking sequence”. In accordance with one embodiment the blocking sequence comprises one or more stop codons that prevent expression of the gene. The site-specific recombinase system is utilized to excise the blocking sequence and thus allow the expression of the gene in a controlled manner.
A number of different site-specific recombinase systems can be used, including but not limited to the Cre/lox system of bacteriophage P1, the FLP/FRT system of yeast, the Gin recombinase of phage Mu, the Pin recombinase of E. coli, and the R/RS system of the pSR1 plasmid. The two preferred site-specific recombinase systems are the bacteriophage P1 Cre/lox and the yeast FLP/FRT systems. In these systems a recombinase (Cre or FLP) will interact specifically with its respective site-specific recombination sequence (lox or FRT respectively) to invert or excise the intervening sequences. The sequence for each of these two systems is relatively short (34 bp for lox and 47 bp for FRT). Currently the FLP/FRT system of yeast is the preferred site-specific recombinase system since it normally functions in a eukaryotic organism (yeast), and is well characterized. Applicants have reason to believe that the eukaryotic origin of the FLP/FRT system allows the FLP/FRT system to function more efficiently in eukaryotic cells than the prokaryotic site-specific recombinase systems.
Depending on the orientation of the site-specific recombination sequences, intervening sequences will either be excised or inverted in the presence of the site-specific recombinase. When the site-specific recombination sequences are orientated in opposite directions relative to one another (i.e., inverted repeats) then any intervening sequences will be inverted relative to the other sequences in the genome. However, if the site-specific recombination sequences are orientated in the same direction relative to one another (i.e., direct repeats) any intervening sequences will be deleted upon interaction with the site-specific recombinase.
The FLP/FRT recombinase system has been demonstrated to function efficiently in plant cells. Experiments on the performance of the FLP/FRT system in both maize and rice protoplasts indicates that FRT site structure, and amount of the FLP protein present, affects excision activity. In general, short incomplete FRT sites leads to higher accumulation of excision products than the complete full-length FRT sites. Site-specific recombination systems can catalyze both intra- and intermolecular reactions in maize protoplasts, indicating that the system can be used for DNA excision as well as integration reactions. The recombination reaction is reversible and this reversibility can compromise the efficiency of the reaction in each direction. Altering the structure of the site-specific recombination sequences is one approach to remedying this situation. The site-specific recombination sequence can be mutated in a manner that the product of the recombination reaction is no longer recognized as a substrate for the reverse reaction, thereby stabilizing the excision event.
Another approach to manipulate the system is based on the entropic advantage of a unimolecular (excision) over a bimolecular (integration) reaction. By limiting the expression of the recombinase enzyme, the efficiency of the integrative recombination, the thermodynamically least favored event, can be reduced. Experiments in maize protoplasts indicate higher concentration of the FLP protein increased the efficiency of the excision reaction.
The use of a site-specific recombinase system to control the expression of gene products allows for the commercial production of useful plant products in economical quantities with environmentally acceptable procedures. The invention is based in part on the ability of a recombinase system to excise a sequence of DNA that separates a promoter from the gene of interest. The excision event operably links the promoter to the gene, thus enabling the expression of the gene. The gene can thus be maintained in a silent state (unexpressed) for any number of generations and then activated by excision of the blocking fragment of DNA, at any desired time and in any number of plants, by crossing the plants containing the “blocked” gene of interest with a plant expressing the site-specific recombinase (FLP, Cre, etc.).
In accordance with one embodiment the disclosed method allows a plant to express one or more genes encoding natural products in concentrations that are deleterious to normal plant or seed development (e.g., a fatty acid or an animal peptide hormone). Alternatively, the present method also allows a plant to express one or more genes encoding foreign products that are deleterious at even low concentrations to normal plant or seed development (e.g., a plastic molecule). Finally, the present invention also allows a plant to express one or more genes which alter normal plant metabolism or development in such a way that the changes are harmful to normal plant or seed development (e.g., inhibit embryo formation, seed germination), and allows a hybrid plant to reproduce by a means that is different from its parent lines (e.g., apomictic versus sexual, and vice versa).